User:David Jung/BCHM3981 RTP Tus - Revision history

David Jung at 06:58, 23 May 2011

David Jung — Mon, 23 May 2011 06:58:27 GMT

←Older revision		Revision as of 06:58, 23 May 2011
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	[[Image:Fig 2.jpg \|thumb\|left\|upright=1.5\|alt= Fig 2. Alignment of the symmetrical RTP B site (''sRB'') (black) with the A (blue) and B (green) sites of the consensus ''Ter'' site DNA (5′–3′ strand only). The region where A and B sites overlap is colored gold. The positions where the ''sRB'' differs from the consensus sequence (indicated by asterisks) and the base specific contacts made by RTP.C110S (indicated by arrows) are highlighted. The ''TerI'' site from B. subtilis 168, uponwhich the whole terminator complex was modeled, has also been aligned.\|Fig 2. Alignment of the symmetrical RTP B site (''sRB'') (black) with the A (blue) and B (green) sites of the consensus ''Ter'' site DNA (5′–3′ strand only). The region where A and B sites overlap is colored gold. The positions where the ''sRB'' differs from the consensus sequence (indicated by asterisks) and the base specific contacts made by RTP.C110S (indicated by arrows) are highlighted. The ''TerI'' site from B. subtilis 168, uponwhich the whole terminator complex was modeled, has also been aligned.]]		[[Image:Fig 2.jpg \|thumb\|left\|upright=1.5\|alt= Fig 2. Alignment of the symmetrical RTP B site (''sRB'') (black) with the A (blue) and B (green) sites of the consensus ''Ter'' site DNA (5′–3′ strand only). The region where A and B sites overlap is colored gold. The positions where the ''sRB'' differs from the consensus sequence (indicated by asterisks) and the base specific contacts made by RTP.C110S (indicated by arrows) are highlighted. The ''TerI'' site from B. subtilis 168, uponwhich the whole terminator complex was modeled, has also been aligned.\|Fig 2. Alignment of the symmetrical RTP B site (''sRB'') (black) with the A (blue) and B (green) sites of the consensus ''Ter'' site DNA (5′–3′ strand only). The region where A and B sites overlap is colored gold. The positions where the ''sRB'' differs from the consensus sequence (indicated by asterisks) and the base specific contacts made by RTP.C110S (indicated by arrows) are highlighted. The ''TerI'' site from B. subtilis 168, uponwhich the whole terminator complex was modeled, has also been aligned.]]
	B. subtilis possesses 9 ''Ter'' sites (''TerI – IX''), 4 able to terminate the clockwise replication fork and 5 able to terminate the anticlockwise replication fork (Fig 1). Each ''Ter'' sites are 30 bp sequences consisting of two 16-17 bp imperfect inverted repeats called the A site and B site that overlap at a fairly conserved trinucleotide sequence (Lewis ''et al.'', 1990) (Fig 2). Each A and B half-site binds a dimer of RTP and fork arrest only occurs when the fork approaches from the B site end (Smith and Wake, 1992).		B. subtilis possesses 9 ''Ter'' sites (''TerI – IX''), 4 able to terminate the clockwise replication fork and 5 able to terminate the anticlockwise replication fork (Fig 1). Each ''Ter'' sites are 30 bp sequences consisting of two 16-17 bp imperfect inverted repeats called the A site and B site that overlap at a fairly conserved trinucleotide sequence (Lewis ''et al.'', 1990) (Fig 2). Each A and B half-site binds a dimer of RTP and fork arrest only occurs when the fork approaches from the B site end (Smith and Wake, 1992).
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	Kralicek ''et al.'' proposed that polar fork arrest arises as a consequence of the two RTP-DNA half-sites having different conformations; which result from different RTP-DNA contacts at the asymmetrical half-sites, as well as interactions between the RTP dimers on the DNA (Kralicek ''et al.'', 1997) (Fig 8). The sequential binding of two RTP dimers to each half-site bends and underwinds the DNA additively (Kralicek ''et al.'', 1997). In Vivian ''et al.''’s X-ray crystallography work on RTP.C110S: ''nRB'' complexes, they observed underwinding of the B site’s upstream end, which suggests that the upstream wing may be involved in contacting the A site to support bending of the DNA (Vivian ''et al.'', 2007). The bending of DNA is thought to facilitate the two RTP dimer’s interactions. Mutagenesis have shown that residues on the β3-strand and β1 loop are crucial in cooperative binding at the A site (Manna ''et al.'', 1996b).		Kralicek ''et al.'' proposed that polar fork arrest arises as a consequence of the two RTP-DNA half-sites having different conformations; which result from different RTP-DNA contacts at the asymmetrical half-sites, as well as interactions between the RTP dimers on the DNA (Kralicek ''et al.'', 1997) (Fig 8). The sequential binding of two RTP dimers to each half-site bends and underwinds the DNA additively (Kralicek ''et al.'', 1997). In Vivian ''et al.''’s X-ray crystallography work on RTP.C110S: ''nRB'' complexes, they observed underwinding of the B site’s upstream end, which suggests that the upstream wing may be involved in contacting the A site to support bending of the DNA (Vivian ''et al.'', 2007). The bending of DNA is thought to facilitate the two RTP dimer’s interactions. Mutagenesis have shown that residues on the β3-strand and β1 loop are crucial in cooperative binding at the A site (Manna ''et al.'', 1996b).
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David Jung at 06:50, 23 May 2011

David Jung — Mon, 23 May 2011 06:50:58 GMT

←Older revision		Revision as of 06:50, 23 May 2011
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	[[Image:Fig 3.jpg \|thumb\|left\|upright=1.3\|alt= Fig 3. Overall structure of the RTP.C110S:nRB complex. “Side view” and “top view” of RTP.C110S in complex with the nRB oligonucleotide. The “wing-up” and “wing-down” monomers are shown in blue and green respectively. The adjacent symmetry-related molecules are represented in grey. The α3-helix are shown to be inserted into the major grooves of DNA.\|Fig 3. Overall structure of the RTP.C110S:nRB complex. “Side view” and “top view” of RTP.C110S in complex with the nRB oligonucleotide. The “wing-up” and “wing-down” monomers are shown in blue and green respectively. The adjacent symmetry-related molecules are represented in grey. The α3-helix are shown to be inserted into the major grooves of DNA.]]		[[Image:Fig 3.jpg \|thumb\|left\|upright=1.3\|alt= Fig 3. Overall structure of the RTP.C110S:nRB complex. “Side view” and “top view” of RTP.C110S in complex with the nRB oligonucleotide. The “wing-up” and “wing-down” monomers are shown in blue and green respectively. The adjacent symmetry-related molecules are represented in grey. The α3-helix are shown to be inserted into the major grooves of DNA.\|Fig 3. Overall structure of the RTP.C110S:nRB complex. “Side view” and “top view” of RTP.C110S in complex with the nRB oligonucleotide. The “wing-up” and “wing-down” monomers are shown in blue and green respectively. The adjacent symmetry-related molecules are represented in grey. The α3-helix are shown to be inserted into the major grooves of DNA.]]

-	<Structure load='2EFW' size='300' frame='true' align='right' caption='RTP' scene='Insert optional scene name here' />	+	<Structure load='2EFW' size='300' frame='true' align='right' caption='2 RTP dimers bound to Ter half sites' scene='Insert optional scene name here' />

	The classical winged-helix motif consists of an αβααββ topology where the “wing” is a loop between the β2-β3 strands. Each RTP monomer comprises of a modified winged-helix motif. It has the β1-strand replaced by a loop and an additional α4 helix following the β3-strand (Vivian ''et al.'', 2007) (Fig 3). The α4 helix acts as a dimerisation interface, forming an antiparallel coiled coil with α4 helix of the other monomer (Vivian ''et al.'', 2007). The α3 helix is the recognition helix and is involved in the specific binding with the DNA sequences via the major groove (Vivian ''et al.'', 2007) (Fig 3, 4). The wings are involved in both protein:proteins and protein:DNA contacts (Manna ''et al.'', 1996b; Pai ''et al.'', 1996). Initially, the RTP dimer was considered to be symmetrical, however this was due the the symmetrical nature of the artificial Ter sequence (''sRB'') used in the study (Wilce ''et al.'', 2001) (Fig 2). Later, Vivian ''et al.'' was able to demonstrate that the RTP dimer is in fact slightly asymmetrical when bound to the native RTP ''TerI'' B site (''nRB'') (Fig 5a) with the greatest deviation in the β1-loop and the wing regions (Fig 5b) (Vivian ''et al.'', 2007). Thus the monomers were differentiated as “wing-up” (upstream) and “wing-down” (downstream) (Fig 3).		The classical winged-helix motif consists of an αβααββ topology where the “wing” is a loop between the β2-β3 strands. Each RTP monomer comprises of a modified winged-helix motif. It has the β1-strand replaced by a loop and an additional α4 helix following the β3-strand (Vivian ''et al.'', 2007) (Fig 3). The α4 helix acts as a dimerisation interface, forming an antiparallel coiled coil with α4 helix of the other monomer (Vivian ''et al.'', 2007). The α3 helix is the recognition helix and is involved in the specific binding with the DNA sequences via the major groove (Vivian ''et al.'', 2007) (Fig 3, 4). The wings are involved in both protein:proteins and protein:DNA contacts (Manna ''et al.'', 1996b; Pai ''et al.'', 1996). Initially, the RTP dimer was considered to be symmetrical, however this was due the the symmetrical nature of the artificial Ter sequence (''sRB'') used in the study (Wilce ''et al.'', 2001) (Fig 2). Later, Vivian ''et al.'' was able to demonstrate that the RTP dimer is in fact slightly asymmetrical when bound to the native RTP ''TerI'' B site (''nRB'') (Fig 5a) with the greatest deviation in the β1-loop and the wing regions (Fig 5b) (Vivian ''et al.'', 2007). Thus the monomers were differentiated as “wing-up” (upstream) and “wing-down” (downstream) (Fig 3).

-	<applet size='200' frame='true' align='left' caption='RTP monomer' scene='User:David_Jung/BCHM3981_RTP_Tus/Monomer/1'/>	+	<applet size='200' frame='true' align='left' caption='RTP monomer' scene='User:David_Jung/BCHM3981_RTP_Tus/Monomer/2'/>
	<applet size='200' frame='true' align='left' caption='RTP dimer' scene='User:David_Jung/BCHM3981_RTP_Tus/Rtp_dimer/1'/>		<applet size='200' frame='true' align='left' caption='RTP dimer' scene='User:David_Jung/BCHM3981_RTP_Tus/Rtp_dimer/1'/>
	<applet size='200' frame='true' align='left' caption='RTP:sRB complex' scene='User:David_Jung/BCHM3981_RTP_Tus/Rtp-srb_complex/2'/>		<applet size='200' frame='true' align='left' caption='RTP:sRB complex' scene='User:David_Jung/BCHM3981_RTP_Tus/Rtp-srb_complex/2'/>

David Jung at 06:47, 23 May 2011

David Jung — Mon, 23 May 2011 06:47:19 GMT

←Older revision		Revision as of 06:47, 23 May 2011
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	[[Image:Fig 2.jpg \|thumb\|left\|upright=1.5\|alt= Fig 2. Alignment of the symmetrical RTP B site (''sRB'') (black) with the A (blue) and B (green) sites of the consensus ''Ter'' site DNA (5′–3′ strand only). The region where A and B sites overlap is colored gold. The positions where the ''sRB'' differs from the consensus sequence (indicated by asterisks) and the base specific contacts made by RTP.C110S (indicated by arrows) are highlighted. The ''TerI'' site from B. subtilis 168, uponwhich the whole terminator complex was modeled, has also been aligned.\|Fig 2. Alignment of the symmetrical RTP B site (''sRB'') (black) with the A (blue) and B (green) sites of the consensus ''Ter'' site DNA (5′–3′ strand only). The region where A and B sites overlap is colored gold. The positions where the ''sRB'' differs from the consensus sequence (indicated by asterisks) and the base specific contacts made by RTP.C110S (indicated by arrows) are highlighted. The ''TerI'' site from B. subtilis 168, uponwhich the whole terminator complex was modeled, has also been aligned.]]		[[Image:Fig 2.jpg \|thumb\|left\|upright=1.5\|alt= Fig 2. Alignment of the symmetrical RTP B site (''sRB'') (black) with the A (blue) and B (green) sites of the consensus ''Ter'' site DNA (5′–3′ strand only). The region where A and B sites overlap is colored gold. The positions where the ''sRB'' differs from the consensus sequence (indicated by asterisks) and the base specific contacts made by RTP.C110S (indicated by arrows) are highlighted. The ''TerI'' site from B. subtilis 168, uponwhich the whole terminator complex was modeled, has also been aligned.\|Fig 2. Alignment of the symmetrical RTP B site (''sRB'') (black) with the A (blue) and B (green) sites of the consensus ''Ter'' site DNA (5′–3′ strand only). The region where A and B sites overlap is colored gold. The positions where the ''sRB'' differs from the consensus sequence (indicated by asterisks) and the base specific contacts made by RTP.C110S (indicated by arrows) are highlighted. The ''TerI'' site from B. subtilis 168, uponwhich the whole terminator complex was modeled, has also been aligned.]]
	B. subtilis possesses 9 ''Ter'' sites (''TerI – IX''), 4 able to terminate the clockwise replication fork and 5 able to terminate the anticlockwise replication fork (Fig 1). Each ''Ter'' sites are 30 bp sequences consisting of two 16-17 bp imperfect inverted repeats called the A site and B site that overlap at a fairly conserved trinucleotide sequence (Lewis ''et al.'', 1990) (Fig 2). Each A and B half-site binds a dimer of RTP and fork arrest only occurs when the fork approaches from the B site end (Smith and Wake, 1992).		B. subtilis possesses 9 ''Ter'' sites (''TerI – IX''), 4 able to terminate the clockwise replication fork and 5 able to terminate the anticlockwise replication fork (Fig 1). Each ''Ter'' sites are 30 bp sequences consisting of two 16-17 bp imperfect inverted repeats called the A site and B site that overlap at a fairly conserved trinucleotide sequence (Lewis ''et al.'', 1990) (Fig 2). Each A and B half-site binds a dimer of RTP and fork arrest only occurs when the fork approaches from the B site end (Smith and Wake, 1992).
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	[[Image:Fig 4.2.jpg \|thumb\|left\|upright=2.0\|alt= Fig 4.2 Summary of the protein:DNA contacts in the RTP.C110S:nRB complex. Phosphate groups (circles), sugar group (pentagons) and the bases (singe letter abbreviation) are shown. Nucleotides in contact with RTP are are in red. Broken lines indicate interactions including hydrogen bonds (red); non-bonded contacts (black) and water-mediated interactions (blue). (a) Contacts between the nRB oligonucleotide and the wing-up half of the RTP.C110S dimer. (b) Contacts between the nRB oligonucleotide and the wing-down half of the RTP.C110S dimer.\|Fig 4.2 Summary of the protein:DNA contacts in the RTP.C110S:nRB complex. Phosphate groups (circles), sugar group (pentagons) and the bases (singe letter abbreviation) are shown. Nucleotides in contact with RTP are are in red. Broken lines indicate interactions including hydrogen bonds (red); non-bonded contacts (black) and water-mediated interactions (blue). (a) Contacts between the nRB oligonucleotide and the wing-up half of the RTP.C110S dimer. (b) Contacts between the nRB oligonucleotide and the wing-down half of the RTP.C110S dimer.]]		[[Image:Fig 4.2.jpg \|thumb\|left\|upright=2.0\|alt= Fig 4.2 Summary of the protein:DNA contacts in the RTP.C110S:nRB complex. Phosphate groups (circles), sugar group (pentagons) and the bases (singe letter abbreviation) are shown. Nucleotides in contact with RTP are are in red. Broken lines indicate interactions including hydrogen bonds (red); non-bonded contacts (black) and water-mediated interactions (blue). (a) Contacts between the nRB oligonucleotide and the wing-up half of the RTP.C110S dimer. (b) Contacts between the nRB oligonucleotide and the wing-down half of the RTP.C110S dimer.\|Fig 4.2 Summary of the protein:DNA contacts in the RTP.C110S:nRB complex. Phosphate groups (circles), sugar group (pentagons) and the bases (singe letter abbreviation) are shown. Nucleotides in contact with RTP are are in red. Broken lines indicate interactions including hydrogen bonds (red); non-bonded contacts (black) and water-mediated interactions (blue). (a) Contacts between the nRB oligonucleotide and the wing-up half of the RTP.C110S dimer. (b) Contacts between the nRB oligonucleotide and the wing-down half of the RTP.C110S dimer.]]
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	Kralicek ''et al.'' proposed that polar fork arrest arises as a consequence of the two RTP-DNA half-sites having different conformations; which result from different RTP-DNA contacts at the asymmetrical half-sites, as well as interactions between the RTP dimers on the DNA (Kralicek ''et al.'', 1997) (Fig 8). The sequential binding of two RTP dimers to each half-site bends and underwinds the DNA additively (Kralicek ''et al.'', 1997). In Vivian ''et al.''’s X-ray crystallography work on RTP.C110S: ''nRB'' complexes, they observed underwinding of the B site’s upstream end, which suggests that the upstream wing may be involved in contacting the A site to support bending of the DNA (Vivian ''et al.'', 2007). The bending of DNA is thought to facilitate the two RTP dimer’s interactions. Mutagenesis have shown that residues on the β3-strand and β1 loop are crucial in cooperative binding at the A site (Manna ''et al.'', 1996b).		Kralicek ''et al.'' proposed that polar fork arrest arises as a consequence of the two RTP-DNA half-sites having different conformations; which result from different RTP-DNA contacts at the asymmetrical half-sites, as well as interactions between the RTP dimers on the DNA (Kralicek ''et al.'', 1997) (Fig 8). The sequential binding of two RTP dimers to each half-site bends and underwinds the DNA additively (Kralicek ''et al.'', 1997). In Vivian ''et al.''’s X-ray crystallography work on RTP.C110S: ''nRB'' complexes, they observed underwinding of the B site’s upstream end, which suggests that the upstream wing may be involved in contacting the A site to support bending of the DNA (Vivian ''et al.'', 2007). The bending of DNA is thought to facilitate the two RTP dimer’s interactions. Mutagenesis have shown that residues on the β3-strand and β1 loop are crucial in cooperative binding at the A site (Manna ''et al.'', 1996b).
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David Jung at 06:39, 23 May 2011

David Jung — Mon, 23 May 2011 06:39:09 GMT

←Older revision		Revision as of 06:39, 23 May 2011
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	[[Image:Fig 4.2.jpg \|thumb\|left\|upright=2.0\|alt= Fig 4.2 Summary of the protein:DNA contacts in the RTP.C110S:nRB complex. Phosphate groups (circles), sugar group (pentagons) and the bases (singe letter abbreviation) are shown. Nucleotides in contact with RTP are are in red. Broken lines indicate interactions including hydrogen bonds (red); non-bonded contacts (black) and water-mediated interactions (blue). (a) Contacts between the nRB oligonucleotide and the wing-up half of the RTP.C110S dimer. (b) Contacts between the nRB oligonucleotide and the wing-down half of the RTP.C110S dimer.\|Fig 4.2 Summary of the protein:DNA contacts in the RTP.C110S:nRB complex. Phosphate groups (circles), sugar group (pentagons) and the bases (singe letter abbreviation) are shown. Nucleotides in contact with RTP are are in red. Broken lines indicate interactions including hydrogen bonds (red); non-bonded contacts (black) and water-mediated interactions (blue). (a) Contacts between the nRB oligonucleotide and the wing-up half of the RTP.C110S dimer. (b) Contacts between the nRB oligonucleotide and the wing-down half of the RTP.C110S dimer.]]		[[Image:Fig 4.2.jpg \|thumb\|left\|upright=2.0\|alt= Fig 4.2 Summary of the protein:DNA contacts in the RTP.C110S:nRB complex. Phosphate groups (circles), sugar group (pentagons) and the bases (singe letter abbreviation) are shown. Nucleotides in contact with RTP are are in red. Broken lines indicate interactions including hydrogen bonds (red); non-bonded contacts (black) and water-mediated interactions (blue). (a) Contacts between the nRB oligonucleotide and the wing-up half of the RTP.C110S dimer. (b) Contacts between the nRB oligonucleotide and the wing-down half of the RTP.C110S dimer.\|Fig 4.2 Summary of the protein:DNA contacts in the RTP.C110S:nRB complex. Phosphate groups (circles), sugar group (pentagons) and the bases (singe letter abbreviation) are shown. Nucleotides in contact with RTP are are in red. Broken lines indicate interactions including hydrogen bonds (red); non-bonded contacts (black) and water-mediated interactions (blue). (a) Contacts between the nRB oligonucleotide and the wing-up half of the RTP.C110S dimer. (b) Contacts between the nRB oligonucleotide and the wing-down half of the RTP.C110S dimer.]]
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	Kralicek ''et al.'' proposed that polar fork arrest arises as a consequence of the two RTP-DNA half-sites having different conformations; which result from different RTP-DNA contacts at the asymmetrical half-sites, as well as interactions between the RTP dimers on the DNA (Kralicek ''et al.'', 1997) (Fig 8). The sequential binding of two RTP dimers to each half-site bends and underwinds the DNA additively (Kralicek ''et al.'', 1997). In Vivian ''et al.''’s X-ray crystallography work on RTP.C110S: ''nRB'' complexes, they observed underwinding of the B site’s upstream end, which suggests that the upstream wing may be involved in contacting the A site to support bending of the DNA (Vivian ''et al.'', 2007). The bending of DNA is thought to facilitate the two RTP dimer’s interactions. Mutagenesis have shown that residues on the β3-strand and β1 loop are crucial in cooperative binding at the A site (Manna ''et al.'', 1996b).		Kralicek ''et al.'' proposed that polar fork arrest arises as a consequence of the two RTP-DNA half-sites having different conformations; which result from different RTP-DNA contacts at the asymmetrical half-sites, as well as interactions between the RTP dimers on the DNA (Kralicek ''et al.'', 1997) (Fig 8). The sequential binding of two RTP dimers to each half-site bends and underwinds the DNA additively (Kralicek ''et al.'', 1997). In Vivian ''et al.''’s X-ray crystallography work on RTP.C110S: ''nRB'' complexes, they observed underwinding of the B site’s upstream end, which suggests that the upstream wing may be involved in contacting the A site to support bending of the DNA (Vivian ''et al.'', 2007). The bending of DNA is thought to facilitate the two RTP dimer’s interactions. Mutagenesis have shown that residues on the β3-strand and β1 loop are crucial in cooperative binding at the A site (Manna ''et al.'', 1996b).
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David Jung at 06:37, 23 May 2011

David Jung — Mon, 23 May 2011 06:37:44 GMT

←Older revision		Revision as of 06:37, 23 May 2011
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		+	== Mechanism ==
		+	[[Image:Fig 8.jpg \|thumb\|right\|upright=2.0\|alt= Fig 8. Models for the generation of RTP–terminator complexes with polar fork arrest activity. The central trinucleotide overlap of the A and B sites of TerI is shown as a hatched box and the dots represent the centres of the flanking 13 bp half site. RTP is represented as shaded boxes of various shapes (conformations). (A) In the induced conformational change (ICC) model, a RTP dimer binds at the B site and bends the DNA to induce cooperative binding of a second dimer at the A site. Binding of the second dimer causes additional remodelling of the Ter site, possibly mediated by protein–protein interactions, which results in a RTP-Ter complex configured for impeding the helicase. (B) In the differential binding affinity (DBA) model, the first dimer binds with relatively high affinity to the B site, but the affinity (thick arrows indicate lower affinities and greater probability of displacement) is still not sufficiently high to impede the replicative helicase. As in the ICC model, the first dimer to the B site alters the DNA to induce cooperative binding of the second dimer at the A site. This results in conformational changes in the DNA and possibly the protein. The affinity of the B site RTP markedly increased. A replication fork approaching from the B site end is unable to dissociate the tightly bound B site dimer. But the RTP at the A has much lower affinity, so that it can be displaced by the replication fork. This in turn decreases the binding affinity of the RTP dimer at the B site, thus enabling helicase to dissociate it as well.\|Fig 8. Models for the generation of RTP–terminator complexes with polar fork arrest activity. The central trinucleotide overlap of the A and B sites of TerI is shown as a hatched box and the dots represent the centres of the flanking 13 bp half site. RTP is represented as shaded boxes of various shapes (conformations). (A) In the induced conformational change (ICC) model, a RTP dimer binds at the B site and bends the DNA to induce cooperative binding of a second dimer at the A site. Binding of the second dimer causes additional remodelling of the Ter site, possibly mediated by protein–protein interactions, which results in a RTP-Ter complex configured for impeding the helicase. (B) In the differential binding affinity (DBA) model, the first dimer binds with relatively high affinity to the B site, but the affinity (thick arrows indicate lower affinities and greater probability of displacement) is still not sufficiently high to impede the replicative helicase. As in the ICC model, the first dimer to the B site alters the DNA to induce cooperative binding of the second dimer at the A site. This results in conformational changes in the DNA and possibly the protein. The affinity of the B site RTP markedly increased. A replication fork approaching from the B site end is unable to dissociate the tightly bound B site dimer. But the RTP at the A has much lower affinity, so that it can be displaced by the replication fork. This in turn decreases the binding affinity of the RTP dimer at the B site, thus enabling helicase to dissociate it as well.]]

		+	It appears that cooperative binding at both A and B sites are required for fork arrest. It has been demonstrated that binding at B site is a pre-requisite to A site binding (Langley ''et al.'', 1993) and binding of B site alone on a vector is not sufficient to stop the advancing helicase (Smith ''et al.'', 1996; Smith ''et al.'', 1994; Smith and Wake, 1992). Only when both half-sites are filled (K<sub>D</sub> = 3 x 10<sup>-11</sup> M) that fork arrest occurs (Duggin ''et al.'', 1999).
		+	Many hypotheses and models have been proposed by a number of researchers to explain this polar mechanism for replication termination (Duggin ''et al.'', 2005; Kralicek ''et al.'', 1997; Manna ''et al.'', 1996a; Manna ''et al.'', 1996b; Wake, 1997). Overall, there have been two models: the differential binding affinity model and the induced conformational change model, both proposed by Kralicek ''et al.'' in 1997 (Kralicek ''et al.'', 1997).



		+	[[Image:Fig 6.jpg \|thumb\|left\|upright=1.0\|alt= RTP fusion proteins have normal binding affinity at ''TerI''. Polyacrylamide gel mobility shift assays were carried out using <sup>32</sup>P-labelled ''TerI'' fragment (0.5 pM). Concentrations (pM) of RTP or RTP fusion proteins are above each lane. The number of dimers bound to the TerI framents is indicated by 0, 1 and 2. The V band is the other non-TerI containing fragment derived from the pID2 plasmid.\|RTP fusion proteins have normal binding affinity at ''TerI''. Polyacrylamide gel mobility shift assays were carried out using <sup>32</sup>P-labelled ''TerI'' fragment (0.5 pM). Concentrations (pM) of RTP or RTP fusion proteins are above each lane. The number of dimers bound to the TerI framents is indicated by 0, 1 and 2. The V band is the other non-TerI containing fragment derived from the pID2 plasmid.]]

-		+	[[Image:Fig 7.jpg \|thumb\|left\|upright=1.0\|alt= RTP fusion proteins have partially replication fork arrest activity in ''B. subtilis''. (a) A Southern blot showing in vivo replication fork arrest assays for the indicated RTP fusion proteins at ''TerI'' on pID2. (b) Normalized fork arrest activity of each RTP fusion protein. Data are shown as the means (± standard errors).\|RTP fusion proteins have partially replication fork arrest activity in ''B. subtilis''. (a) A Southern blot showing in vivo replication fork arrest assays for the indicated RTP fusion proteins at ''TerI'' on pID2. (b) Normalized fork arrest activity of each RTP fusion protein. Data are shown as the means (± standard errors).]]
-	~~== Mechanism ==~~	+
-	It appears that cooperative binding at both A and B sites are required for fork arrest. It has been demonstrated that binding at B site is a pre-requisite to A site binding (Langley ''et al.'', 1993) and binding of B site alone on a vector is not sufficient to stop the advancing helicase (Smith ''et al.'', 1996; Smith ''et al.'', 1994; Smith and Wake, 1992). Only when both half-sites are filled (K<sub>D</sub> = 3 x 10<sup>-11</sup> M) that fork arrest occurs (Duggin ''et al.'', 1999).	+
-	Many hypotheses and models have been proposed by a number of researchers to explain this polar mechanism for replication termination (Duggin ''et al.'', 2005; Kralicek ''et al.'', 1997; Manna ''et al.'', 1996a; Manna ''et al.'', 1996b; Wake, 1997). Overall, there have been two models: the differential binding affinity model and the induced conformational change model, both proposed by Kralicek ''et al.'' in 1997 (Kralicek ''et al.'', 1997).	+
-		+
-	[[Image:Fig 8.jpg \|thumb\|~~right~~\|upright=2.0\|alt= ~~Fig 8. Models for the generation of RTP–terminator complexes with polar~~ fork arrest activity~~. The central trinucleotide overlap of the A and~~ B ~~sites of TerI is shown as a hatched box and the dots represent the centres of the flanking 13 bp half site~~. ~~RTP is represented as shaded boxes of various shapes (conformations)~~. (~~A) In the induced conformational change (ICC) model,~~ a RTP dimer binds at the B site and bends the DNA to induce cooperative binding of a second dimer at the A site. Binding of the second dimer causes additional remodelling of the Ter site, possibly mediated by protein–protein interactions, which results in a RTP-Ter complex configured for impeding the helicase. (B) In the differential binding affinity (DBA) model, the first dimer binds with relatively high affinity to the B site, but the affinity (thick arrows indicate lower affinities and greater probability of displacement) is still not sufficiently high to impede the replicative helicase. As in the ICC model, the first dimer to the B site alters the DNA to induce cooperative binding of the second dimer at the A ~~site. This results~~ in ~~conformational changes in the DNA and possibly the protein. The affinity of the B site RTP markedly increased. A~~ replication fork ~~approaching from the B site end is unable to dissociate the tightly bound B site dimer. But~~ the RTP at ~~the A has much lower affinity, so that it can be displaced by the replication fork~~. ~~This in turn decreases the binding affinity of the RTP dimer at the B site, thus enabling helicase to dissociate it as well.\|Fig 8. Models for the generation of RTP–terminator complexes with polar~~ fork arrest activity. ~~The central trinucleotide overlap of the A and B sites of TerI is~~ shown as ~~a hatched box and~~ the ~~dots represent the centres of the flanking 13 bp half site. RTP is represented as shaded boxes of various shapes~~ (~~conformations~~). ~~(A) In the induced conformational change (ICC) model, a~~ RTP ~~dimer binds at the~~ B ~~site and bends the DNA to induce cooperative binding of a second dimer at the A site~~. ~~Binding of the second dimer causes additional remodelling of the Ter site, possibly mediated by protein–protein interactions, which results in a RTP-Ter complex configured for impeding the helicase~~. (B) In the differential binding affinity (DBA) model, the first dimer binds with relatively high affinity to the B site, but the affinity (thick arrows indicate lower affinities and greater probability of displacement) is still not sufficiently high to impede the replicative helicase. As in the ICC model, the first dimer to the B site alters the DNA to induce cooperative binding of the second dimer at the A ~~site. This results~~ in ~~conformational changes in the DNA and possibly the protein. The affinity of the B site RTP markedly increased. A~~ replication fork ~~approaching from the B site end is unable to dissociate the tightly bound B site dimer. But~~ the RTP at ~~the A has much lower affinity, so that it can be displaced by the replication~~ fork~~. This in turn decreases the binding affinity~~ of ~~the~~ RTP ~~dimer at~~ the ~~B site, thus enabling helicase to dissociate it as well~~.]]	+

	'''Differential binding affinity model'''		'''Differential binding affinity model'''
Line 150:		Line 150:
	Kralicek ''et al.'' proposed that polar fork arrest arises as a consequence of the two RTP-DNA half-sites having different conformations; which result from different RTP-DNA contacts at the asymmetrical half-sites, as well as interactions between the RTP dimers on the DNA (Kralicek ''et al.'', 1997) (Fig 8). The sequential binding of two RTP dimers to each half-site bends and underwinds the DNA additively (Kralicek ''et al.'', 1997). In Vivian ''et al.''’s X-ray crystallography work on RTP.C110S: ''nRB'' complexes, they observed underwinding of the B site’s upstream end, which suggests that the upstream wing may be involved in contacting the A site to support bending of the DNA (Vivian ''et al.'', 2007). The bending of DNA is thought to facilitate the two RTP dimer’s interactions. Mutagenesis have shown that residues on the β3-strand and β1 loop are crucial in cooperative binding at the A site (Manna ''et al.'', 1996b).		Kralicek ''et al.'' proposed that polar fork arrest arises as a consequence of the two RTP-DNA half-sites having different conformations; which result from different RTP-DNA contacts at the asymmetrical half-sites, as well as interactions between the RTP dimers on the DNA (Kralicek ''et al.'', 1997) (Fig 8). The sequential binding of two RTP dimers to each half-site bends and underwinds the DNA additively (Kralicek ''et al.'', 1997). In Vivian ''et al.''’s X-ray crystallography work on RTP.C110S: ''nRB'' complexes, they observed underwinding of the B site’s upstream end, which suggests that the upstream wing may be involved in contacting the A site to support bending of the DNA (Vivian ''et al.'', 2007). The bending of DNA is thought to facilitate the two RTP dimer’s interactions. Mutagenesis have shown that residues on the β3-strand and β1 loop are crucial in cooperative binding at the A site (Manna ''et al.'', 1996b).

-	~~{{multiple image~~	+
-	~~\| align = left~~	+
-	~~\| direction = horizontal~~	+
-	~~\| footer = RTP fusion proteins have partially replication fork arrest activity in ''B. subtilis'' despite having normal binding affinity for Ter sites.~~	+
-	~~\| image1 = Fig 6.jpg~~	+
-	\| alt1 = Polyacrylamide gel mobility shift assays were carried out using <sup>32</sup>P-labelled ''TerI'' fragment (0.5 pM). Concentrations (pM) of RTP or RTP fusion proteins are above each lane. The number of dimers bound to the TerI framents is indicated by 0, 1 and 2. The V band is the other non-TerI containing fragment derived from the pID2 plasmid.	+
-	\| caption1 = Polyacrylamide gel mobility shift assays were carried out using <sup>32</sup>P-labelled ''TerI'' fragment (0.5 pM). Concentrations (pM) of RTP or RTP fusion proteins are above each lane. The number of dimers bound to the TerI framents is indicated by 0, 1 and 2. The V band is the other non-TerI containing fragment derived from the pID2 plasmid.	+
-	~~\| image2 = Fig 7.jpg~~	+
-	\| alt2 = (a) A Southern blot showing in vivo replication fork arrest assays for the indicated RTP fusion proteins at ''TerI'' on pID2. (b) Normalized fork arrest activity of each RTP fusion protein. Data are shown as the means (± standard errors).	+
-	\| caption2 = (a) A Southern blot showing in vivo replication fork arrest assays for the indicated RTP fusion proteins at ''TerI'' on pID2. (b) Normalized fork arrest activity of each RTP fusion protein. Data are shown as the means (± standard errors).	+
-	}}	+

David Jung at 06:22, 23 May 2011

David Jung — Mon, 23 May 2011 06:22:30 GMT

←Older revision		Revision as of 06:22, 23 May 2011
Line 153:		Line 153:
	\| align = left		\| align = left
	\| direction = horizontal		\| direction = horizontal
-	~~\| width = 1350~~	+	\| footer = RTP fusion proteins have partially replication fork arrest activity in ''B. subtilis'' despite having normal binding affinity for Ter sites.
-	\| footer = RTP fusion proteins have partially replication fork arrest activity in ''B. subtilis'' despite having normal binding affinity for Ter sites	+	\| image1 = Fig 6.jpg
-	\| image1 = ~~Image:~~Fig 6.jpg	+
	\| alt1 = Polyacrylamide gel mobility shift assays were carried out using <sup>32</sup>P-labelled ''TerI'' fragment (0.5 pM). Concentrations (pM) of RTP or RTP fusion proteins are above each lane. The number of dimers bound to the TerI framents is indicated by 0, 1 and 2. The V band is the other non-TerI containing fragment derived from the pID2 plasmid.		\| alt1 = Polyacrylamide gel mobility shift assays were carried out using <sup>32</sup>P-labelled ''TerI'' fragment (0.5 pM). Concentrations (pM) of RTP or RTP fusion proteins are above each lane. The number of dimers bound to the TerI framents is indicated by 0, 1 and 2. The V band is the other non-TerI containing fragment derived from the pID2 plasmid.
	\| caption1 = Polyacrylamide gel mobility shift assays were carried out using <sup>32</sup>P-labelled ''TerI'' fragment (0.5 pM). Concentrations (pM) of RTP or RTP fusion proteins are above each lane. The number of dimers bound to the TerI framents is indicated by 0, 1 and 2. The V band is the other non-TerI containing fragment derived from the pID2 plasmid.		\| caption1 = Polyacrylamide gel mobility shift assays were carried out using <sup>32</sup>P-labelled ''TerI'' fragment (0.5 pM). Concentrations (pM) of RTP or RTP fusion proteins are above each lane. The number of dimers bound to the TerI framents is indicated by 0, 1 and 2. The V band is the other non-TerI containing fragment derived from the pID2 plasmid.
-	\| image2 = ~~Image:~~Fig 7.jpg	+	\| image2 = Fig 7.jpg
	\| alt2 = (a) A Southern blot showing in vivo replication fork arrest assays for the indicated RTP fusion proteins at ''TerI'' on pID2. (b) Normalized fork arrest activity of each RTP fusion protein. Data are shown as the means (± standard errors).		\| alt2 = (a) A Southern blot showing in vivo replication fork arrest assays for the indicated RTP fusion proteins at ''TerI'' on pID2. (b) Normalized fork arrest activity of each RTP fusion protein. Data are shown as the means (± standard errors).
	\| caption2 = (a) A Southern blot showing in vivo replication fork arrest assays for the indicated RTP fusion proteins at ''TerI'' on pID2. (b) Normalized fork arrest activity of each RTP fusion protein. Data are shown as the means (± standard errors).		\| caption2 = (a) A Southern blot showing in vivo replication fork arrest assays for the indicated RTP fusion proteins at ''TerI'' on pID2. (b) Normalized fork arrest activity of each RTP fusion protein. Data are shown as the means (± standard errors).

David Jung at 06:18, 23 May 2011

David Jung — Mon, 23 May 2011 06:18:39 GMT

←Older revision		Revision as of 06:18, 23 May 2011
Line 150:		Line 150:
	Kralicek ''et al.'' proposed that polar fork arrest arises as a consequence of the two RTP-DNA half-sites having different conformations; which result from different RTP-DNA contacts at the asymmetrical half-sites, as well as interactions between the RTP dimers on the DNA (Kralicek ''et al.'', 1997) (Fig 8). The sequential binding of two RTP dimers to each half-site bends and underwinds the DNA additively (Kralicek ''et al.'', 1997). In Vivian ''et al.''’s X-ray crystallography work on RTP.C110S: ''nRB'' complexes, they observed underwinding of the B site’s upstream end, which suggests that the upstream wing may be involved in contacting the A site to support bending of the DNA (Vivian ''et al.'', 2007). The bending of DNA is thought to facilitate the two RTP dimer’s interactions. Mutagenesis have shown that residues on the β3-strand and β1 loop are crucial in cooperative binding at the A site (Manna ''et al.'', 1996b).		Kralicek ''et al.'' proposed that polar fork arrest arises as a consequence of the two RTP-DNA half-sites having different conformations; which result from different RTP-DNA contacts at the asymmetrical half-sites, as well as interactions between the RTP dimers on the DNA (Kralicek ''et al.'', 1997) (Fig 8). The sequential binding of two RTP dimers to each half-site bends and underwinds the DNA additively (Kralicek ''et al.'', 1997). In Vivian ''et al.''’s X-ray crystallography work on RTP.C110S: ''nRB'' complexes, they observed underwinding of the B site’s upstream end, which suggests that the upstream wing may be involved in contacting the A site to support bending of the DNA (Vivian ''et al.'', 2007). The bending of DNA is thought to facilitate the two RTP dimer’s interactions. Mutagenesis have shown that residues on the β3-strand and β1 loop are crucial in cooperative binding at the A site (Manna ''et al.'', 1996b).

		+	{{multiple image
		+	\| align = left
		+	\| direction = horizontal
		+	\| width = 1350
		+	\| footer = RTP fusion proteins have partially replication fork arrest activity in ''B. subtilis'' despite having normal binding affinity for Ter sites
		+	\| image1 = Image:Fig 6.jpg
		+	\| alt1 = Polyacrylamide gel mobility shift assays were carried out using <sup>32</sup>P-labelled ''TerI'' fragment (0.5 pM). Concentrations (pM) of RTP or RTP fusion proteins are above each lane. The number of dimers bound to the TerI framents is indicated by 0, 1 and 2. The V band is the other non-TerI containing fragment derived from the pID2 plasmid.
		+	\| caption1 = Polyacrylamide gel mobility shift assays were carried out using <sup>32</sup>P-labelled ''TerI'' fragment (0.5 pM). Concentrations (pM) of RTP or RTP fusion proteins are above each lane. The number of dimers bound to the TerI framents is indicated by 0, 1 and 2. The V band is the other non-TerI containing fragment derived from the pID2 plasmid.
		+	\| image2 = Image:Fig 7.jpg
		+	\| alt2 = (a) A Southern blot showing in vivo replication fork arrest assays for the indicated RTP fusion proteins at ''TerI'' on pID2. (b) Normalized fork arrest activity of each RTP fusion protein. Data are shown as the means (± standard errors).
		+	\| caption2 = (a) A Southern blot showing in vivo replication fork arrest assays for the indicated RTP fusion proteins at ''TerI'' on pID2. (b) Normalized fork arrest activity of each RTP fusion protein. Data are shown as the means (± standard errors).
		+	}}

David Jung at 06:08, 23 May 2011

David Jung — Mon, 23 May 2011 06:08:04 GMT

←Older revision		Revision as of 06:08, 23 May 2011
Line 139:		Line 139:
	Many hypotheses and models have been proposed by a number of researchers to explain this polar mechanism for replication termination (Duggin ''et al.'', 2005; Kralicek ''et al.'', 1997; Manna ''et al.'', 1996a; Manna ''et al.'', 1996b; Wake, 1997). Overall, there have been two models: the differential binding affinity model and the induced conformational change model, both proposed by Kralicek ''et al.'' in 1997 (Kralicek ''et al.'', 1997).		Many hypotheses and models have been proposed by a number of researchers to explain this polar mechanism for replication termination (Duggin ''et al.'', 2005; Kralicek ''et al.'', 1997; Manna ''et al.'', 1996a; Manna ''et al.'', 1996b; Wake, 1997). Overall, there have been two models: the differential binding affinity model and the induced conformational change model, both proposed by Kralicek ''et al.'' in 1997 (Kralicek ''et al.'', 1997).

		+	[[Image:Fig 8.jpg \|thumb\|right\|upright=2.0\|alt= Fig 8. Models for the generation of RTP–terminator complexes with polar fork arrest activity. The central trinucleotide overlap of the A and B sites of TerI is shown as a hatched box and the dots represent the centres of the flanking 13 bp half site. RTP is represented as shaded boxes of various shapes (conformations). (A) In the induced conformational change (ICC) model, a RTP dimer binds at the B site and bends the DNA to induce cooperative binding of a second dimer at the A site. Binding of the second dimer causes additional remodelling of the Ter site, possibly mediated by protein–protein interactions, which results in a RTP-Ter complex configured for impeding the helicase. (B) In the differential binding affinity (DBA) model, the first dimer binds with relatively high affinity to the B site, but the affinity (thick arrows indicate lower affinities and greater probability of displacement) is still not sufficiently high to impede the replicative helicase. As in the ICC model, the first dimer to the B site alters the DNA to induce cooperative binding of the second dimer at the A site. This results in conformational changes in the DNA and possibly the protein. The affinity of the B site RTP markedly increased. A replication fork approaching from the B site end is unable to dissociate the tightly bound B site dimer. But the RTP at the A has much lower affinity, so that it can be displaced by the replication fork. This in turn decreases the binding affinity of the RTP dimer at the B site, thus enabling helicase to dissociate it as well.\|Fig 8. Models for the generation of RTP–terminator complexes with polar fork arrest activity. The central trinucleotide overlap of the A and B sites of TerI is shown as a hatched box and the dots represent the centres of the flanking 13 bp half site. RTP is represented as shaded boxes of various shapes (conformations). (A) In the induced conformational change (ICC) model, a RTP dimer binds at the B site and bends the DNA to induce cooperative binding of a second dimer at the A site. Binding of the second dimer causes additional remodelling of the Ter site, possibly mediated by protein–protein interactions, which results in a RTP-Ter complex configured for impeding the helicase. (B) In the differential binding affinity (DBA) model, the first dimer binds with relatively high affinity to the B site, but the affinity (thick arrows indicate lower affinities and greater probability of displacement) is still not sufficiently high to impede the replicative helicase. As in the ICC model, the first dimer to the B site alters the DNA to induce cooperative binding of the second dimer at the A site. This results in conformational changes in the DNA and possibly the protein. The affinity of the B site RTP markedly increased. A replication fork approaching from the B site end is unable to dissociate the tightly bound B site dimer. But the RTP at the A has much lower affinity, so that it can be displaced by the replication fork. This in turn decreases the binding affinity of the RTP dimer at the B site, thus enabling helicase to dissociate it as well.]]

	'''Differential binding affinity model'''		'''Differential binding affinity model'''
Line 148:		Line 149:

	Kralicek ''et al.'' proposed that polar fork arrest arises as a consequence of the two RTP-DNA half-sites having different conformations; which result from different RTP-DNA contacts at the asymmetrical half-sites, as well as interactions between the RTP dimers on the DNA (Kralicek ''et al.'', 1997) (Fig 8). The sequential binding of two RTP dimers to each half-site bends and underwinds the DNA additively (Kralicek ''et al.'', 1997). In Vivian ''et al.''’s X-ray crystallography work on RTP.C110S: ''nRB'' complexes, they observed underwinding of the B site’s upstream end, which suggests that the upstream wing may be involved in contacting the A site to support bending of the DNA (Vivian ''et al.'', 2007). The bending of DNA is thought to facilitate the two RTP dimer’s interactions. Mutagenesis have shown that residues on the β3-strand and β1 loop are crucial in cooperative binding at the A site (Manna ''et al.'', 1996b).		Kralicek ''et al.'' proposed that polar fork arrest arises as a consequence of the two RTP-DNA half-sites having different conformations; which result from different RTP-DNA contacts at the asymmetrical half-sites, as well as interactions between the RTP dimers on the DNA (Kralicek ''et al.'', 1997) (Fig 8). The sequential binding of two RTP dimers to each half-site bends and underwinds the DNA additively (Kralicek ''et al.'', 1997). In Vivian ''et al.''’s X-ray crystallography work on RTP.C110S: ''nRB'' complexes, they observed underwinding of the B site’s upstream end, which suggests that the upstream wing may be involved in contacting the A site to support bending of the DNA (Vivian ''et al.'', 2007). The bending of DNA is thought to facilitate the two RTP dimer’s interactions. Mutagenesis have shown that residues on the β3-strand and β1 loop are crucial in cooperative binding at the A site (Manna ''et al.'', 1996b).
		+
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David Jung at 04:55, 23 May 2011

David Jung — Mon, 23 May 2011 04:55:22 GMT

←Older revision		Revision as of 04:55, 23 May 2011
Line 8:		Line 8:
	[[Image:Fig 2.jpg \|thumb\|left\|upright=1.5\|alt= Fig 2. Alignment of the symmetrical RTP B site (''sRB'') (black) with the A (blue) and B (green) sites of the consensus ''Ter'' site DNA (5′–3′ strand only). The region where A and B sites overlap is colored gold. The positions where the ''sRB'' differs from the consensus sequence (indicated by asterisks) and the base specific contacts made by RTP.C110S (indicated by arrows) are highlighted. The ''TerI'' site from B. subtilis 168, uponwhich the whole terminator complex was modeled, has also been aligned.\|Fig 2. Alignment of the symmetrical RTP B site (''sRB'') (black) with the A (blue) and B (green) sites of the consensus ''Ter'' site DNA (5′–3′ strand only). The region where A and B sites overlap is colored gold. The positions where the ''sRB'' differs from the consensus sequence (indicated by asterisks) and the base specific contacts made by RTP.C110S (indicated by arrows) are highlighted. The ''TerI'' site from B. subtilis 168, uponwhich the whole terminator complex was modeled, has also been aligned.]]		[[Image:Fig 2.jpg \|thumb\|left\|upright=1.5\|alt= Fig 2. Alignment of the symmetrical RTP B site (''sRB'') (black) with the A (blue) and B (green) sites of the consensus ''Ter'' site DNA (5′–3′ strand only). The region where A and B sites overlap is colored gold. The positions where the ''sRB'' differs from the consensus sequence (indicated by asterisks) and the base specific contacts made by RTP.C110S (indicated by arrows) are highlighted. The ''TerI'' site from B. subtilis 168, uponwhich the whole terminator complex was modeled, has also been aligned.\|Fig 2. Alignment of the symmetrical RTP B site (''sRB'') (black) with the A (blue) and B (green) sites of the consensus ''Ter'' site DNA (5′–3′ strand only). The region where A and B sites overlap is colored gold. The positions where the ''sRB'' differs from the consensus sequence (indicated by asterisks) and the base specific contacts made by RTP.C110S (indicated by arrows) are highlighted. The ''TerI'' site from B. subtilis 168, uponwhich the whole terminator complex was modeled, has also been aligned.]]
	B. subtilis possesses 9 ''Ter'' sites (''TerI – IX''), 4 able to terminate the clockwise replication fork and 5 able to terminate the anticlockwise replication fork (Fig 1). Each ''Ter'' sites are 30 bp sequences consisting of two 16-17 bp imperfect inverted repeats called the A site and B site that overlap at a fairly conserved trinucleotide sequence (Lewis ''et al.'', 1990) (Fig 2). Each A and B half-site binds a dimer of RTP and fork arrest only occurs when the fork approaches from the B site end (Smith and Wake, 1992).		B. subtilis possesses 9 ''Ter'' sites (''TerI – IX''), 4 able to terminate the clockwise replication fork and 5 able to terminate the anticlockwise replication fork (Fig 1). Each ''Ter'' sites are 30 bp sequences consisting of two 16-17 bp imperfect inverted repeats called the A site and B site that overlap at a fairly conserved trinucleotide sequence (Lewis ''et al.'', 1990) (Fig 2). Each A and B half-site binds a dimer of RTP and fork arrest only occurs when the fork approaches from the B site end (Smith and Wake, 1992).
		+
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		+
		+
		+
		+
		+
		+	== Structure ==
		+	[[Image:Fig 3.jpg \|thumb\|left\|upright=1.3\|alt= Fig 3. Overall structure of the RTP.C110S:nRB complex. “Side view” and “top view” of RTP.C110S in complex with the nRB oligonucleotide. The “wing-up” and “wing-down” monomers are shown in blue and green respectively. The adjacent symmetry-related molecules are represented in grey. The α3-helix are shown to be inserted into the major grooves of DNA.\|Fig 3. Overall structure of the RTP.C110S:nRB complex. “Side view” and “top view” of RTP.C110S in complex with the nRB oligonucleotide. The “wing-up” and “wing-down” monomers are shown in blue and green respectively. The adjacent symmetry-related molecules are represented in grey. The α3-helix are shown to be inserted into the major grooves of DNA.]]
		+
		+	<Structure load='2EFW' size='300' frame='true' align='right' caption='RTP' scene='Insert optional scene name here' />
		+
		+	The classical winged-helix motif consists of an αβααββ topology where the “wing” is a loop between the β2-β3 strands. Each RTP monomer comprises of a modified winged-helix motif. It has the β1-strand replaced by a loop and an additional α4 helix following the β3-strand (Vivian ''et al.'', 2007) (Fig 3). The α4 helix acts as a dimerisation interface, forming an antiparallel coiled coil with α4 helix of the other monomer (Vivian ''et al.'', 2007). The α3 helix is the recognition helix and is involved in the specific binding with the DNA sequences via the major groove (Vivian ''et al.'', 2007) (Fig 3, 4). The wings are involved in both protein:proteins and protein:DNA contacts (Manna ''et al.'', 1996b; Pai ''et al.'', 1996). Initially, the RTP dimer was considered to be symmetrical, however this was due the the symmetrical nature of the artificial Ter sequence (''sRB'') used in the study (Wilce ''et al.'', 2001) (Fig 2). Later, Vivian ''et al.'' was able to demonstrate that the RTP dimer is in fact slightly asymmetrical when bound to the native RTP ''TerI'' B site (''nRB'') (Fig 5a) with the greatest deviation in the β1-loop and the wing regions (Fig 5b) (Vivian ''et al.'', 2007). Thus the monomers were differentiated as “wing-up” (upstream) and “wing-down” (downstream) (Fig 3).
		+
		+	<applet size='200' frame='true' align='left' caption='RTP monomer' scene='User:David_Jung/BCHM3981_RTP_Tus/Monomer/1'/>
		+	<applet size='200' frame='true' align='left' caption='RTP dimer' scene='User:David_Jung/BCHM3981_RTP_Tus/Rtp_dimer/1'/>
		+	<applet size='200' frame='true' align='left' caption='RTP:sRB complex' scene='User:David_Jung/BCHM3981_RTP_Tus/Rtp-srb_complex/2'/>
		+
		+	[[Image:Fig 4.1.jpg \|thumb\|left\|upright=1.6\|alt= Fig 4.1 ''TerI'' site from ''B. subtilis'' showing the nRB sequence (blue)\|Fig 4.1 ''TerI'' site from ''B. subtilis'' showing the nRB sequence (blue)]]
		+
		+	[[Image:Fig 5.jpg \|thumb\|upright=1.6\|alt= Fig 5. Structural differences between the RTP.C110S:nRB and RTP.C110S:sRB complexes. (a) RTP.C110S crystal structures from nRB (blue) and sRB (yellow) complexes superimposed upon one another (from “top view”). The angle between α2 and α3 is indicated for each subunit. (b) Plot of root mean square deviation (RMSD) values versus residue number for each α-carbon position of the two RTP.C110S:nRB monomers (wing-up and wing-down) compared with each other (green) and with the RTP.C110S:sRB monomer structure (wing-up, red and wing-down, black).\|Fig 5. Structural differences between the RTP.C110S:nRB and RTP.C110S:sRB complexes. (a) RTP.C110S crystal structures from nRB (blue) and sRB (yellow) complexes superimposed upon one another (from “top view”). The angle between α2 and α3 is indicated for each subunit. (b) Plot of root mean square deviation (RMSD) values versus residue number for each α-carbon position of the two RTP.C110S:nRB monomers (wing-up and wing-down) compared with each other (green) and with the RTP.C110S:sRB monomer structure (wing-up, red and wing-down, black).]]
		+
		+	[[Image:Fig 4.2.jpg \|thumb\|left\|upright=2.0\|alt= Fig 4.2 Summary of the protein:DNA contacts in the RTP.C110S:nRB complex. Phosphate groups (circles), sugar group (pentagons) and the bases (singe letter abbreviation) are shown. Nucleotides in contact with RTP are are in red. Broken lines indicate interactions including hydrogen bonds (red); non-bonded contacts (black) and water-mediated interactions (blue). (a) Contacts between the nRB oligonucleotide and the wing-up half of the RTP.C110S dimer. (b) Contacts between the nRB oligonucleotide and the wing-down half of the RTP.C110S dimer.\|Fig 4.2 Summary of the protein:DNA contacts in the RTP.C110S:nRB complex. Phosphate groups (circles), sugar group (pentagons) and the bases (singe letter abbreviation) are shown. Nucleotides in contact with RTP are are in red. Broken lines indicate interactions including hydrogen bonds (red); non-bonded contacts (black) and water-mediated interactions (blue). (a) Contacts between the nRB oligonucleotide and the wing-up half of the RTP.C110S dimer. (b) Contacts between the nRB oligonucleotide and the wing-down half of the RTP.C110S dimer.]]
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-	== Structure ==
-	<Structure load='2EFW' size='300' frame='true' align='right' caption='RTP' scene='Insert optional scene name here' />

-	The classical winged-helix motif consists of an αβααββ topology where the “wing” is a loop between the β2-β3 strands. Each RTP monomer comprises of a modified winged-helix motif. It has the β1-strand replaced by a loop and an additional α4 helix following the β3-strand (Vivian ''et al.'', 2007) (Fig 3). The α4 helix acts as a dimerisation interface, forming an antiparallel coiled coil with α4 helix of the other monomer (Vivian ''et al.'', 2007). The α3 helix is the recognition helix and is involved in the specific binding with the DNA sequences via the major groove (Vivian ''et al.'', 2007) (Fig 3, 4). The wings are involved in both protein:proteins and protein:DNA contacts (Manna ''et al.'', 1996b; Pai ''et al.'', 1996). Initially, the RTP dimer was considered to be symmetrical, however this was due the the symmetrical nature of the artificial Ter sequence (''sRB'') used in the study (Wilce ''et al.'', 2001) (Fig 2). Later, Vivian ''et al.'' was able to demonstrate that the RTP dimer is in fact slightly asymmetrical when bound to the native RTP ''TerI'' B site (''nRB'') (Fig 5a) with the greatest deviation in the β1-loop and the wing regions (Fig 5b) (Vivian ''et al.'', 2007). Thus the monomers were differentiated as “wing-up” (upstream) and “wing-down” (downstream) (Fig 3).

-	<applet size='200' frame='true' align='left' caption='RTP monomer' scene='User:David_Jung/BCHM3981_RTP_Tus/Monomer/1'/>
-	<applet size='200' frame='true' align='left' caption='RTP dimer' scene='User:David_Jung/BCHM3981_RTP_Tus/Rtp_dimer/1'/>
-	<applet size='200' frame='true' align='left' caption='RTP:sRB complex' scene='User:David_Jung/BCHM3981_RTP_Tus/Rtp-srb_complex/2'/>

David Jung at 04:40, 23 May 2011

David Jung — Mon, 23 May 2011 04:40:58 GMT

←Older revision		Revision as of 04:40, 23 May 2011
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-	'''The Replication Terminator Protein (RTP)''' is a protein involved in termination of replication in the gram positive bacterium, ''Bacillus subtilis''. RTP was first identified in 1989, showing analogous function to Tus protein present in ''Escherichia coli'' (Lewis~~, Smith and Wake,~~ 1989). Both RTP and Tus bind to termination sites (''Ter'' sequences) present in the bacterial chromosome, terminating replication. Polar directionality of termination is assumed as circular bacterial chromosome is replicated bidirectionally with one replication fork going clockwise and the other one going anticlockwise. The RTP-''Ter'' complex must therefore ~~block a~~ replication fork ~~coming~~ from ~~one side but permit a~~ fork from the ~~opposite side~~. This is demonstrated in the ''Escherichia coli'' counterpart due to its apparent asymmetric structure.	+	'''The Replication Terminator Protein (RTP)''' is a protein involved in termination of replication in the gram positive bacterium, Bacillus subtilis. RTP was first identified in 1989, showing analogous function to Tus protein present in Escherichia coli (Lewis ''et al.'' 1989). Both RTP and Tus bind to termination sites (''Ter'' sequences) present in the bacterial chromosome, terminating replication. Polar directionality of termination is assumed as circular bacterial chromosome is replicated bidirectionally with one replication fork going clockwise and the other one going anticlockwise. The RTP-''Ter'' complex must therefore allow the replication fork to pass unimpeded from the “permissive” direction, while halting the fork from the “blocking” direction (Griffiths, Andersen, and Wake, 1998). This is demonstrated in the ''Escherichia coli'' counterpart due to its apparent asymmetric structure (Kamada ''et al.'', 1996). However, this asymmetry is not observed in RTP's structure.
-		+

	Even though the protein is known to be involved in replication termination, its biological function is not well understood as mutants that lack certain ''Ter'' sites are shown to be viable ''in vitro'' (Iismaa and Wake. 1987). Thus, the biological functions of replication terminator proteins in bacteria have long been speculated. There are two hypotheses: inhibition of production of multimeric DNA, and post-initiation control of replication. Multimeric DNA is a dsDNA where multiple copies of the whole sequence are present. It has been shown, in the case of Tus in ''Escherichia coli'', that without Tus-Ter interaction, it is more prone to overreplication (Hiasa and Marians, 1994). This is thought to be due to lack of inhibition of movement of DnaB, a helicase, along the replication fork by Tus-Ter complex. It was also shown that replication terminator protein is involved in post-initiation control of replication. The first level of control of replication was thought to occur before initiation. However, it has been experimentally determined that RTP-Ter complex maybe involved in second level of control after initiation to inhibit overreplication (Henckes ''et al.,'' 1989).		Even though the protein is known to be involved in replication termination, its biological function is not well understood as mutants that lack certain ''Ter'' sites are shown to be viable ''in vitro'' (Iismaa and Wake. 1987). Thus, the biological functions of replication terminator proteins in bacteria have long been speculated. There are two hypotheses: inhibition of production of multimeric DNA, and post-initiation control of replication. Multimeric DNA is a dsDNA where multiple copies of the whole sequence are present. It has been shown, in the case of Tus in ''Escherichia coli'', that without Tus-Ter interaction, it is more prone to overreplication (Hiasa and Marians, 1994). This is thought to be due to lack of inhibition of movement of DnaB, a helicase, along the replication fork by Tus-Ter complex. It was also shown that replication terminator protein is involved in post-initiation control of replication. The first level of control of replication was thought to occur before initiation. However, it has been experimentally determined that RTP-Ter complex maybe involved in second level of control after initiation to inhibit overreplication (Henckes ''et al.,'' 1989).